The BCL-2 family of proteins regulates apoptosis, a critical physiologic process that maintains tissue homeostasis by eliminating injured or unwanted cells. MCL-1 is an anti-apoptotic BCL-2 protein whose overexpression is linked to the pathogenesis and chemoresistance of human cancer. MCL-1 neutralizes pro-apoptotic BCL-2 family members, including NOXA, PUMA, BIM, and BAK, thus ensuring cell survival. Pharmacological disruption of specific BCL-2 family protein interactions can induce apoptosis in cancer cells, but selective targeting of MCL-1 has remained out of reach. The goal of this project is to develop a structurally stable, cell permeable, and highly selective MCL-1 targeting BH3 ligand with which to study the biology of MCL-1 and evaluate the therapeutic efficacy of targeting MCL-1 in cancer. The BCL-2 homology domain 3 (BH3) mediates essential protein interactions among BCL-2 family proteins. To broadly explore the targeting capacity of BCL-2 family BH3 domains for MCL-1, a panel of Stabilized Alpha-Helix of BCL-2 domains (SAHBs) will be designed, synthesized, and tested for selective binding affinity for MCL-1 among the BCL-2 family anti-apoptotic proteins. The Walensky laboratory has demonstrated that SAHBs can recapitulate the native a-helical structure of BH3 death domains and readily penetrate intact cells by an endocytic mechanism. To examine the biochemical and physiological effects of targeting MCL-1, a battery of in vitro and cellular assays will be performed using selective SAHB inhibitors of MCL-1 (SIMs). First, to assess the capacity of SIMs to target MCL-1 and dissociate its heterodimeric interactions with pro-apoptotic BCL-2 proteins such as BAK, competitive fluorescence polarization binding assays and in situ co-immunoprecipitation studies will be performed. Induction of BAK-mediated mitochondrial apoptosis by SIMs will be examined in cytochrome c release assays performed on isolated mouse liver mitochondria. The ability of SIMs to reactivate apoptosis alone and in combination with other pro-apoptotic stimuli will be evaluated in MCL-1-dependent cancer cell lines by monitoring cell viability and caspase 3/7 activation. Finally, the therapeutic efficacy of SIMs alone and in combination with other pro- apoptotic agents will be investigated in xenograft models of MCL-1 dependent cancers. The anti-apoptotic BCL-2 family protein MCL-1 has emerged as a major resistance factor in relapsed and refractory cancer. To overcome the challenge of reactivating apoptosis in MCL-1 overexpressing cancers, a novel chemical biology approach will be deployed to develop selective MCL-1 inhibitors for use as molecular probes to study the biology of MCL-1 and as prototype therapeutics to combat MCL-1 driven cancer.